1. Notes

2. Result Statistics

Figure 1. False discovery rate (FDR) curve. X axis is the number of peptide-spectrum matches (PSM) being kept. Y axis is the corresponding FDR.

Figure 2. PSM score distribution. (a) Distribution of PEAKS peptide score; (b) Scatterplot of PEAKS peptide score versus precursor mass error.

(a)

(b)

Figure 3. De novo result validation. Distribution of residue local confidence: (a) Residues in de novo sequences validated by confident database peptide assignment; (b) Residues in "de novo only" sequences.

(a)

(b)

Table 1. Statistics of data. # of MS scans 8833 # of MS/MS scans 176637 Table 2. Result filtration parameters. Peptide -10lgP ≥15 Peptide Ascore ≥0 Protein -10lgP ≥23 Proteins unique peptides ≥2 De novo ALC Score ≥50% Table 3. Statistics of filtered result. Peptide-Spectrum Matches 47021 Peptide sequences 19379 Protein groups 861 Proteins 861 Proteins (#Unique Peptides) 677 (>2); 184 (=2); 0 (=1); FDR (Peptide-Spectrum Matches) 12.6% FDR (Peptide Sequences) 26.5% FDR (Protein) 43.7% De Novo Only Spectra 25265

Table 4. PTM profile. Name ∆Mass Position #PSM -10lgP Area AScore Carbamidomethyl 57.02 C 4364 200.00 2.07E6 1000.00 Methylation(KR) 14.02 KR 3851 124.97 7.44E5 18.61 Deamidation .98 NQ 3126 200.00 3.92E5 17.01 Oxidation 15.99 M 1825 160.33 1.01E6 1000.00 Methylation(others) 14.02 DEHST 1482 145.77 3.1E5 11.06 Pyro-glu from Q -17.03 N-term 765 154.80 5.96E5 1000.00 Dehydration -18.01 DSTY,C-term 589 89.94 9.43E4 0.00 Acetylation 42.01 Protein N-term 572 175.49 1.69E5 1000.00 Acetylation 42.01 N-term 561 97.51 4.46E5 1000.00 Phosphorylation 79.97 STY 425 112.97 1.31E5 0.00 Acetylation 42.01 K 361 100.71 1.02E5 157.67 Didehydro -2.02 STY,C-term 325 94.89   0.00 Carbamylation 43.01 K,N-term 313 76.43   90.97 Dihydroxy 31.99 FKPRWY 231 98.89 5.34E4 90.79 Amidation -.98 C-term 208 55.99   1000.00 Sodium 21.98 DE,C-term 192 68.41 2.13E5 0.00 Dimethylation(KR) 28.03 KR 178 86.49 4.03E5 71.25 Formylation 27.99 K,N-term 166 42.29 7.71E4 70.36 Dethiomethyl -48.00 M 117 72.63 1.89E5 1000.00 Carboxylation 43.99 DKW 97 60.62 3.45E4 38.32 Ethyl 28.03 DE,N-term,C-term 91 89.03 1.08E5 0.00 Lysaminoadipicsealde -1.03 K 89 81.17 3.59E4 1000.00 Oxidation 15.99 HW 86 80.55 1.48E5 1000.00 Deamidation .98 R 83 96.55 1.27E5 3.38 Biotin 226.08 K,N-term 79 39.72   0.00 Ammonia loss -17.03 N 77 81.60 7.08E4 1000.00 O-Diisopropylphosphate 164.06 KSTY,N-term 66 68.73 1.01E5 0.00 Carboxymethyl 58.01 C 62 80.20 1.17E5 1000.00 Sulfation 79.96 STY 57 61.94 2.87E5 17.01 Hexose 162.05 NSY 54 48.88 3.11E5 1000.00 Amidine 41.03 K,N-term 44 56.91   30.64 Guanidination 42.02 K 42 149.58 1.46E5 276.80 Diethylation 56.06 K,N-term 31 76.88 5.62E4 207.12 MethylamineST 13.03 ST 29 51.56   18.01 Cation:Li 6.01 DE,C-term 29 82.92   15.43 Propionamide 71.04 K,N-term 26 43.64 1.15E5 49.83 Methyl+Deamidated 15.00 NQ 25 66.42 6.05E4 22.05 Ubiquitin 114.04 KST 25 60.29 1.31E5 28.36 Carboxylation 43.99 E 22 77.57 1.41E5 11.10 Methylation(C-term) 14.02 C-term 22 56.18 6.05E5 1000.00 Pyro-glu from E -18.01 N-term 21 80.73 8.1E4 1000.00 Met->Aha -4.99 M 21 57.03 1.56E5 1000.00 Cation:K 37.96 DE,C-term 18 39.57 1.21E5 0.00 EtOH 44.03 KR 12 34.90 9.18E4 13.38 Sulphone 31.99 M 11 56.65 3.73E5 1000.00 PEITC 163.05 K,N-term 10 56.51 2.37E5 12.28 Ammonium 17.03 DE,C-term 10 55.12 1.55E5 19.39 Acetyl 42.01 ST 9 28.61   9.40 SMA 127.06 N-term 9 34.51   88.88 Oxidation 15.99 DKNPR 8 40.41 4.44E4 0.00 Propionald+40 40.03 HK 8 46.56 1.39E5 37.14 Deoxy -15.99 DST 8 63.68   46.87 Im_biotin 225.09 N-term 8 44.58 2.18E5 69.82 HNE 156.12 HK 8 58.48 1.85E5 126.79 Aminotyrosine 15.01 Y 8 46.22 2.97E5 1000.00 ISD_z+2_ion -15.01 N-term 8 19.25   1000.00 Propargylamine 37.03 DE,C-term 8 32.99 5.79E4 85.10 LeuArgGlyGly 383.23 K 7 52.12 2.84E5 1000.00 LG-pyrrole 316.20 N-term 7 36.35 2.15E5 52.58 Cation:Ni[II] 55.92 DE 7 39.64   14.04 MercaptoEthanol 60.00 S 7 31.08   5.47 Formylation 27.99 Protein N-term 7 21.96 2.09E5 1000.00 Kynurenin 3.99 W 6 37.64 2.01E5 1000.00 Iodination 125.90 Y 6 77.80 1.37E5 17.01 MG-H1 54.01 R 6 27.10   6.65 Pyrrole 78.05 K 6 35.86 5.65E4 122.77 Carboxymethyl 58.01 K,N-term 6 32.95 1.81E4 64.77 MDA54 54.01 K 6 30.30 2.09E5 0.00 azole -20.03 S 6 42.68 1.46E4 43.47 Myristoylation 210.20 CK,N-term 6 53.30 1.7E4 1000.00 ethylamino 27.05 ST 5 65.75 4.24E5 7.03 Crotonyl 68.03 K 5 18.74   18.00 Carboxyethyl 72.02 K 5 33.50 1.15E5 1000.00 Ammonia loss -17.03 Protein N-term 5 44.61   1000.00 NeuAc 291.10 ST 5 43.65   14.04 Pyridylacetyl 119.04 K,N-term 5 32.29 5.96E5 67.12 BITC 149.03 K,N-term 5 43.50 1.06E5 1000.00 EtOH 44.03 C 5 34.56 2.42E5 1000.00 CAMthiopropanoyl 145.02 K 5 37.99   80.99 LG-anhyropyrrole 298.19 N-term 4 17.81 8.19E4 1000.00 p-guanosine 345.05 K 4 22.05   0.00 NMM 111.03 K 4 31.92 7.03E3 24.44 AEC-MAEC 59.02 ST 4 63.80 1.77E5 7.77 O-Ethylphosphate 108.00 K,N-term 4 26.64 2.52E5 14.62 B-methylthiol 45.99 DN 4 16.78 2.58E4 28.13 NDA 175.04 K,N-term 4 23.45 7.96E4 19.27 Acetald+26 26.02 HK 4 59.87 3.43E4 23.70 Octanoyl 126.10 ST 4 19.85   0.00 Ethanolamine 43.04 DE,C-term 4 44.76 5.63E4 16.76 Cytopiloyne 362.14 K,N-term 4 44.28 6.02E4 0.00 DSP 88.00 N-term 4 24.24 1.99E5 1000.00 Arg->Npo 80.99 R 4 22.50 8.38E5 46.57 Lipoyl 188.03 K 4 34.38   56.47 Carbofuran 58.03 S 4 34.21 1.2E5 1000.00 Phosphopropargyl 117.00 T 4 25.85 1.57E5 1000.00

3. Experiment Control

Figure 4. Precursor mass error of peptide-spectrum matches (PSM) in filtered result. (a) Distribution of precursor mass error in ppm; (b) Scatterplot of precursor m/z versus precursor mass error in ppm.

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(b)

Table 5. Number of identified peptides in each sample by the number of missed cleavages

Missed Cleavages 0 1 2 3 4+ Virulento 10923 6221 1822 413 0

4. Other Information

Table 6. Search parameters. Search Engine Name: PEAKS Parent Mass Error Tolerance: 0.07 Da Fragment Mass Error Tolerance: 0.07 Da Precursor Mass Search Type: monoisotopic Enzyme: Trypsin Max Missed Cleavages: 3 Non-specific Cleavage: none Fixed Modifications:   Carbamidomethylation: 57.02 Variable Modifications:   Oxidation (M): 15.99   Acetylation (K): 42.01   Acetylation (Protein N-term): 42.01   Acetylation (N-term): 42.01   Amidation: -0.98   Beta-methylthiolation: 45.99   Biotinylation: 226.08   Carbamylation: 43.01   and 304 more... Max Variable PTM Per Peptide: 3 Database: Paracoccidioides_Pb18 Taxon: All Searched Entry: 7000 FDR Estimation: Enabled De novo score (ALC%) threshold: 15 Peptide hit threshold (-10logP): 30.0 Peaks run ID: 13 Different data refine parameters are used for this search:

Table 7. Instrument parameters. Fractions: VEV_1_3_RA4_01_1235.d, VEV_2_3_RA5_01_1236.d, VEV_3_ 3_RA6_01_1237.d Ion Source: ESI(nano-spray) Fragmentation Mode: CID, CAD(y and b ions) MS Scan Mode: Quadrupole MS/MS Scan Mode: Time of Flight (TOF)